Forced Degradation Study of Monoclonal Antibody Using Two- Dimensional Liquid Chromatography

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, terminal cyclization, aggregation, and fragmentation. Forced degradation study is a common practice to assess the potential modifications and degradation pathways of mAbs upon extreme conditions, including light exposure and extreme local pH. The degraded samples are subject to characterization using a wide array of analyses, including ion exchange chromatography (IEC) for charge variants and size exclusion chromatography (SEC) for size variants. In this proof-of-concept study, a two-dimensional liquid chromatography (2D-LC) approach was successfully applied to mAb forced degradation samples for evaluation of size distribution in the IEC charge profile. The charge variant peaks in first-dimension (1D) IEC were fractioned by a heart-cutting schedule, and the integrated peak parking feature accommodated later-eluting 1D cuts when the previous ones were still under second-dimension (2D) analysis. Thus, for a singly IEC injection, all IEC fractions were acquired by the 2D for SEC analysis. This study demonstrated that 2D SEC was compatible with 1D IEC for 2D-LC analysis. The method sensitivity was sufficient to determine the aggregates and fragments in each individual IEC cut. The calculated total aggregates and fragments in the stressed mAb-1 samples were comparable to those detected by one dimensional SEC. Most importantly, the 2D IEC-SEC approach was capable of bridging the mAb charge and size variants in a real-time and efficient manner. It was observed that aggregates were enriched in the most basic region in the IEC charge profile, especially in the highly degraded samples. This is the first IEC-SEC 2D-LC application for intact antibody analysis as per the authors’ knowledge.